Expression pattern of the transcription factor Olig2in response to brain injuries: Implicationsfor neuronal repairAnnalisa Buffo*†‡, Milan R. Vosko§, Dilek Erturk*, Gerhard F. Hamann§¶, Mathias Jucker�, David Rowitch**,and Magdalena Gotz*‡††

*Institute for Stem Cell Research, National Research Center for Environment and Health, Ingolstadter Landstrasse 1, D-85764 Neuherberg�Munich, Germany;†Department of Neuroscience, University of Turin, Corso Raffaello 30, I-10125 Turin, Italy; §Department of Neurology, Ludwig-Maximilians University,Klinikum Grosshadern, Marchioninistrasse 15, 81377 Munich, Germany; ¶Department of Neurology, Dr. Horst Schmidt Klinik, Ludwig-Erhard-Strasse 100,65199 Wiesbaden, Germany; �Department of Cellular Neurology, Hertie-Institute for Clinical Brain Research, Otfried-Muller Strasse 27, D-72076 Tubingen,Germany; **Department of Pediatric Oncology, Dana–Farber Cancer Institute, Harvard Medical School, Boston, MA 02115; and ††Department of Physiology,Ludwig-Maximilians University, Munich, Pettenkoferstrasse 12, D-80336 Munich, Germany

Edited by Hans Thoenen, Max Planck Institute of Neurobiology, Martinsried, Germany, and approved October 24, 2005 (received for review July 30, 2005)

Despite the presence of neural stem cells and ongoing neurogen-esis in some regions of the adult mammalian brain, neurons are notreplaced in most brain regions after injury. With the aim to unravelfactors contributing to the failure of neurogenesis in the injuredcerebral cortex, we examined the expression of cell fate determi-nants after acute brain injuries, such as stab wound or focalischemia, and in a model of chronic amyloid deposition. Althoughnone of the neurogenic factors, such as Pax6, Mash1, Ngn2, wasdetected in the injured parenchyma, we observed a strong up-regulation of the bHLH transcription factor Olig2, but not Olig1,upon acute and chronic injury. To examine the function of Olig2 inbrain lesion, we injected retroviral vectors containing a dominantnegative form of Olig2 into the lesioned cortex 2 days after a stabwound. Antagonizing Olig2 function resulted in a significant num-ber of infected cells generating immature neurons that were notobserved after injection of the control virus. These data, therefore,imply Olig2 as a repressor of neurogenesis in cells reacting to braininjury and open innovative perspectives toward evoking endoge-nous neuronal repair.

amyloid � gliosis � mouse neocortex � pax6 � stab wound

The discovery that adult neural stem cells in the mammalianbrain continue to generate neurons throughout life has shed

new light on the possibility to repair degenerated neurons byendogenous sources. The similarity of adult neural stem cells toastroglial cells (1, 2) prompts the question whether glial cellsreacting to injury also may have a broader potential. Astrocytesmediate the wound healing reaction of the central nervoussystem (CNS) upon injury (3) and reexpress molecules [vimen-tin, nestin, tenascin-C, and glia-fibrillary acidic protein (GFAP);see refs. 3 and 4] that are absent in mature astrocytes but presentduring development in radial glial cells (5–7) and adult neuralstem cells (8–13). Whereas during development adult neuralstem cells and radial glia generate neurons (1, 2, 6, 14–16),reactive astrocytes do not, despite their partial dedifferentiationresponse. However, immature astrocytes can generate neuronsin vitro upon stimulation with intrinsic or extrinsic factors (17,18). This neurogenic potential is not restricted to astrocytes,because NG2-positive glial precursors also can generate neuronsupon exposure to certain culture conditions (19) or transplan-tation into neurogenic zones in vivo (20) but fail to regenerateneurons upon injury in vivo (21). Thus, endogenous precursorand glial cell populations have the potential to generate neuronsbut fail to do so upon injury in vivo.

Here, we set out to elucidate the molecular cues involved inthe glial fate restriction after injury. We examined transcriptionfactors, such as Pax6, Ngn2, Mash1, Gsh2, Olig1, and Olig2, thatregulate neuron-glia fate decisions during development and are

present in precursors in the subependymal zone (SEZ) of theadult mouse forebrain (22–24). Our results identify Olig2 as akey factor in reaction of glial cells to brain injury and show that,by interfering with Olig2 function, some degree of endogenousneurogenesis can be evoked. Thus, these results show proof ofprinciple evidence that neurogenesis can occur in the mamma-lian neocortex, even in severe injury conditions, such as acutestab-wound lesion.

Materials and MethodsAnimals and Surgical Procedures. Forty-three CD1 mice (2–6months, Charles River Laboratories, Sulzfeld, Germany) wereanesthetized (ketamine, 100 mg�kg, Ketavet; Amersham Phar-macia, Erlangen, Germany, and xylazine, 5 mg�kg, Rompun;Bayer, Leverkusen, Germany) and underwent a stab wound inthe right cerebral neocortex (Bregma from �0.9 mm to �2.7mm, latero-lateral 1.5–2.5 mm) or hindbrain (lateral reticularnucleus, Bregma from �8 to �8.12 mm, 1.1 mm latero-lateral).Mice were killed at 24 h (n � 3) and 3 (n � 6), 7 (n � 9), and30 days after lesion (n � 3; intact, n � 2). In some experiments,1 �l of retroviral suspension containing green fluorescent pro-tein (GFP), Olig2-VP16-IRES-GFP (Olig2VP16), or Pax6-IRES-GFP (titers: 106 to 107, produced as described in ref. 22)was injected 2 days after injury within 100–150 �m from thelesion track at 0.5- to 1-mm depth from the dura (n � 22, survivaltimes: 2, 7, 14, and 30 days after injection).

To examine focal ischemia, seven C57BL�6 male mice (2months old) underwent occlusion of the middle cerebral artery(MCAO) by the intraluminal filament model (25), according toref. 26, for 2 h. Reperfusion was induced by thread withdrawalunder short inhalation anesthesia. The animals were killed after24 h (n � 3) or 7 days (n � 4).

As a model for amyloid plaque deposition, we examined fourpairs of 6- to 9-month-old C57BL�6-TgN (Thy1-APPKM670/671NL;Thy1-PS1L166P) mice (Thy1-APPPS) and wild-type littermates.The transgenic mice were generated by coinjection of Thy1-APPKM670/671NL and Thy1-PS1L166P constructs into B6 oocytes(line 21; R. Radde and M.J., unpublished observations, but seerefs. 27 and 28). The Thy1 promoter restricted transgene ex-pression to CNS neurons. For the present study, line 21 was used.

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: dcx, doublecortin; dpi, days after injection; dpl, days after lesion; GFAP, glialfibrillary acidic protein; GM, gray matter; LM-, lineage marker-negative; MCAO, middlecerebral artery occlusion; Olig2�, Olig2-immunopositive.

‡To whom correspondence may be addressed. E-mail: magdalena.goetz@gsf.de orannalisa.buffo@gsf.de.

© 2005 by The National Academy of Sciences of the USA

www.pnas.org�cgi�doi�10.1073�pnas.0506535102 PNAS � December 13, 2005 � vol. 102 � no. 50 � 18183–18188

NEU

ROSC

IEN

CE

Dow

nloa

ded

by g

uest

on

Janu

ary

24, 2

021

Cerebral amyloidosis (Fig. 5, which is published as supportinginformation on the PNAS web site) developed at the age of 6–8weeks.

Proliferation Analysis and Histological Procedures. The DNA baseanalog 5-bromodeoxyuridine (BrdUrd, Sigma) was injected i.p.(100 mg�kg body weight) 2 h before perfusion to label fastproliferating cells (short pulse). Alternatively, animals receivedBrdUrd in the drinking water (long pulse, 1 mg�ml) from the firstpostsurgery day for either 2 (3 days survival) or 6 days (7 dayssurvival) or 2 weeks (Thy1-APPPS mice). The mice were tran-scardially perfused with 4% paraformaldehyde in phosphatebuffer, and sections were cut after cryoprotection. In MCAO-treated brains, volumetric analysis (25) showed infarction in thebasal ganglia and cortex, corresponding to the territory suppliedby the middle cerebral artery. Immunostainings and in situhybridization were performed according to standard protocols,and the antibodies used are listed in the Supporting Materials,which is published as supporting information on the PNAS website).

Quantitative Analysis. Quantifications (cell densities, marker co-expression and nuclear size; three sections per animal, threeanimals for each time point or lesion model) were performed bymeans of NEUROLUCIDA and NEUROEXPLORER (Microbright-field, Colchester, VT), connected to an Axiophot Zeiss micro-scope (�40 objective). The analysis was performed on 200,000�m2 areas within the cortical gray matter (GM) and localized 100�m away from the stab-lesion track (both laterally and medially)or the edge of the ischemic core, and in corresponding areas ofthe intact hemisphere, wild-type, and Thy1-APPPS cortices. Allresults are presented as averages and standard deviations, unlessindicated differently. Statistical analysis was performed by theStudent or one-sample t test.

ResultsIncrease in Olig2-Immunoreactive Cells in the Cortex After Stab-Wound Injury. After stab-wound injury of the adult cerebralcortex, microglia is the first cell type to react (29), followed byan increase in NG2-positive cells, astroglia reaction (increase innumber and hypertrophy), and proliferation of various cell types(Fig. 6, which is published as supporting information on thePNAS web site; see also Fig. 2I). In this context, we examinedthe transcription factors Pax6, Mash1, Gsh2, Ngn2, Olig1, andOlig2 1–30 days after the stab wound by immunochemistry. NoPax6-, Mash1-, Gsh2- or Ngn2-immunopositive cells were de-tectable in the lesioned cortex despite positive signals in the adultsubependymal zone or the embryonic telencephalon (data notshown), consistent with the absence of even a low degree ofneurogenesis around the lesion as monitored by doublecortin(dcx) staining (data not shown) or retroviral infection (seebelow). In contrast, 3–7 days after the stab wound, we observeda prominent increase in the number of Olig2-immunoreactivecells (spread in the cortex up to 400–500 �m from the lesion) incomparison with the intact contralateral cortex, where Olig2-positive cells were much more rare (Fig. 1 A–C). The increase inOlig2 was also detected at the mRNA level, as revealed by in situhybridization (Fig. 1 D and E), and this reaction returned tocontrol levels 30 days after lesion (dpl, Fig. 1C). Notably, theincrease in Olig2-expressing cells was specific because even theclose family member Olig1 was not affected in its expressionlevels upon stab-wound injury (Fig. 7, which is published assupporting information on the PNAS web site).

Identity of Olig2-Immunopositive (Olig2�) Cells in the Intact andStab-Wound Injured Cortex. Next, we characterized the identity ofcells expressing Olig2 (see Table 1, which is published assupporting information on the PNAS web site, for cell type

identification criteria). In the intact adult CNS, Olig2 is ex-pressed in mature oligodendrocytes and NG2-positive glialprecursors (30–32). Consistent with these data, the majority ofNG2-positive (NG2�) cells in the intact cortical GM (�70%)contained Olig2, but they only accounted for 26% of the Olig2�cell pool (Fig. 2 B, F, and G). Mature CC1-expressing (CC1�)oligodendrocytes and S100�-positive (S100���NG2-�CNPase-,see Table 1) astrocytes also comprised a small proportion ofOlig2� cells (19% and 11%, respectively; Fig. 2 A, C, F, and G,see also ref. 33). The considerable proportion of remainingOlig2� cells was actually negative for all the tested lineagemarkers (referred to as LM-, Fig. 2F) for all tests, includingneuronal, microglial, leukocyte, endothelial markers, nestin,platelet-derived growth factor receptor-�, and Lewis-X (see alsoTable 1). However, virtually all (91 � 3%) Olig2� nuclei wereSox10-immunopositive (Sox10�; Fig. 2D) (34), including theLM- cells, that thus appear to belong to the oligodendrocytelineage. Because the mean nuclear size of Olig2��Sox��LM-cells (52 � 6 �m2) resembles the one of NG2� cells but is notablydistinct from that of mature oligodendrocytes (20 � 6 �m2),these cells seem to represent a precursor stage in the oligoden-drocyte lineage.

Notably, a similar composition of the Olig2� population wasobserved 7 days after a stab wound (Fig. 2F). GFAP is ahallmark of reactive astrocytes expressed in a subset of the totalastrocyte S100�� population after injury. Olig2 was detectedonly in a fraction of the GFAP-positive astrocytes in the injuredcortical GM (10.2 � 1.3% at 3 dpl and 14.65 � 4.6% at 7 dpl)and the GFAP-positive cells constituted only 7–9% of theOlig2� cells (7.1 � 1.4 at 3 dpl and 8.9 � 4.6 at 7 dpl). Thus,

Fig. 1. Increase in the number of Olig2-immunoreactive cells after a stabwound. (A and B) Frontal sections of the gray matter (GM) in the adult mouseneocortex stained for Olig2. Note the increase in the number of Olig2� cellsafter a stab wound (s-w, B; yellow line indicates the lesion site, dashedcontours outline some unspecific�autofluorescent staining, and arrowheadspoint to some Olig2� cells) compared to the intact cortex (A, arrowheadsindicate Olig2� cells) as quantified in the histogram (C). *, statistically signif-icant differences; hs, hours; dpl, days after lesion. In situ hybridization revealsOlig2 mRNA up-regulation after a stab wound (E) compared to the intactcortex (D). (Scale bars: 100 �m.)

18184 � www.pnas.org�cgi�doi�10.1073�pnas.0506535102 Buffo et al.

Dow

nloa

ded

by g

uest

on

Janu

ary

24, 2

021

Olig2 expression was not restricted to the GFAP-expressingreactive astrocytes, but occurred more widespread in astrocytesand NG2� cells. Because the proportion of these glial popula-tions amongst the Olig2� cells remained relatively constant afterthe 3-fold increase in the Olig2� cells after injury, this evidencesuggests that all of these diverse cell types contribute to theincrease in the number of Olig2� cells. As the total number ofS100�� astrocytes and NG2� cells increased after a stab wound(Fig. 6 A–C), the increase in the number of Olig2� cells may bepartially due to cell proliferation.

To characterize the proliferating cell types after a stab wound,we labeled the fast proliferating cells by injecting the DNA baseanalog BrdUrd 2 h before analysis or we supplied BrdUrd forseveral days in the drinking water to label all dividing cells,including those proliferating very slowly (see Materials andMethods). Both paradigms revealed a massive increase in thedensity of BrdUrd-positive cells 3 dpl (12-fold increase, 2-hpulse) that decreased later and reached control levels 4 weeksafter injury (Figs. 2H and 6A). Olig2� cells, which constitutedabout half of all of the fast cycling cells in the intact parenchyma,also comprised up to 60% of the expanded fast dividing cell poolaround the lesion (Fig. 2H), suggesting that they are highlyproliferative and that this proliferation is responsible, at leastpartially, of the increase in Olig2� cells after a stab wound.Notably, hardly any LM- cells were labeled with a short BrdUrdpulse, whereas they did incorporate BrdUrd supplied for severaldays (Fig. 2I), indicating that these cells are slowly proliferating.Consistent with the data presented above, several of the slowlydividing LM- cells were Olig2� (Fig. 2E). In summary, thetranscription factor Olig2 is prominent in diverse sets of fastproliferating glia and slow dividing LM- cells in reaction toinjury.

Lineage Analysis of Proliferating Cells After a Stab Wound. To assessthe cell fate and lineage of the proliferating cells describedabove, we used replication-incompetent retroviral vectors thatincorporate stably into the genome of proliferating cells and

inherit their genome encoding, e.g., for the GFP, to the progenygenerated from the infected precursors (see, e.g., ref. 35). Whenwe injected a retroviral vector containing only GFP (22) in thecortical parenchyma close to the lesion site 2 days after a stabwound (Fig. 3A), infected cells were detected in the gray or whitematter at all examined times after injection (dpi, Fig. 3B). Asexpected, the composition of infected cells analyzed at 2 dpi (Fig.3J) closely resembled the composition of the fast proliferatingcells as determined by BrdUrd-pulse labeling around the time ofviral injection (Fig. 2I). Notably, 54 � 15% of the infected cellswere Olig2� (n � 120) and they largely consisted of NG2�precursors (Fig. 3J), whereas few LM- cells were labeled, in linewith the failure of MLV-based retroviral genomes to incorporateinto the genome of slow proliferating cells (36). However, 1 weekafter virus injection, almost 50% of all GFP-labeled (GFP�)cells were LM- (Fig. 3 F and K), suggesting that these cells derivefrom some of the infected glial cell types, most likely the NG2�cells because they decrease in number when LM- cells increase.When the progeny of infected cells was analyzed at 14 or 30 dpi,the proportion of the LM- cells had decreased again, but nowmostly S100�� astroglial cells had increased in number (Fig.3K). These data suggest that the LM- cells increase as reactionto injury, originating from NG2� cells and later differentiatinginto astrocytes, consistent with a previous analysis with adeno-viral vectors (21). Although these data would be consistent withsome transition between glial lineages, no neurons were gener-ated because none of the infected GFP� cells contained anyneuronal markers (dcx, HuC�D, NeuN) at all of the examinedtime points (Fig. 3K), reconfirming the absence of neurogenesisdescribed above.

Transduction of a Dominant-Negative Form of Olig2 Allows Neuro-genesis. Next, we set out to determine the role of Olig2 in theseproliferating cells after injury. Toward this end, we used retro-viral vectors carrying Olig2 cDNA, where the Olig2 repressordomain was replaced by the herpes simplex VP16 protein andlinked by an IRES domain to GFP (Olig2VP16) (22, 37–39).

Fig. 2. Identification of Olig2-immunoreactive cells after stab-wound and proliferation analysis. (A–E) Frontal sections of the intact neocortical GMimmunostained as indicated. Olig2 immunoractivity was detected in CC1� oligodendrocytes (A), NG2� glial precursors (B), and S100�� astrocytes (C). Arrowspoint to colabeled cells, and arrowheads point to cells devoid of Olig2. (D) Olig2 and Sox10 coexpression (arrows) in the cortical GM are shown. (E) The micrographdepicts Olig2� nuclei triple labeled for BrdUrd (red) and a mixture of antibodies (in blue: NG2, CC1, and S100�) close to the stab-wound track: Severalproliferating Brdu��Olig2� cells are lineage marker-negative (arrow). Histograms in F depict the proportions of cell types in the Olig2� population in the intactcortex and after a stab wound, whereas in G, the percentage of Olig2� cells amongst different glial populations is shown. The fractions of Olig2� astrocytesincreased at 7 dpl (P � 0.012) compared to the intact situation. The opposite was observed for the NG2� cells in comparison with the intact cortex (P � 0.003at 3 days; P � 0.042 at 7 days) and the proportion of Olig2��CC1� oligodendrocytes rose 7 dpl (P � 0.044). (H) Olig2�BrdUrd colabeled cells (indicated also aspercentages) are shown over the density of BrdUrd-positive cells in the intact and stab-lesioned cortex (2-h BrdUrd pulse). (I) The composition of BrdUrd-positivecells is depicted after 2 h (Left) or several days of BrdUrd pulse (Right). (Scale bars: A–C and E, 20 �m; D, 100 �m.)

Buffo et al. PNAS � December 13, 2005 � vol. 102 � no. 50 � 18185

NEU

ROSC

IEN

CE

Dow

nloa

ded

by g

uest

on

Janu

ary

24, 2

021

Previous studies have shown that the VP16 domain exerts effectsspecifically and exclusively in cells expressing Olig2, where itantagonizes the endogenous role of Olig2 (22, 37–39). Becausehalf of all infected cells contained Olig2, this construct shouldaffect Olig2 function in these cells. The phenotype ofOlig2VP16-infected cells at 2 dpi was similar to that after controlvirus injections (data not shown), whereas we observed anobvious decrease in the proportion of the LM- cells 7 days afterOlig2VP16 transduction compared to the control virus (Fig. 3 Kand L). Conversely, the proportion of S100�� cells amongst theGFP� cells was increased upon Olig2VP16 infection comparedto control virus injected cells (Fig. 3 K and L). Most excitingly,a significant proportion of Olig2VP16-infected cells (5 � 2%,n � 228, three mice) acquired neuronal characteristics such asdcx immunoreactivity and an elongated morphology reminiscentof migrating neuroblasts 7 dpi (Fig. 3 G and L). Notably, thisproportion further increased to 12 � 3% at 14 dpi (P � 0.008,n � 320, Fig. 3L), indicating a considerable time required forrespecification toward the neuronal lineage. Consistent with aneurogenic lineage redirection, we also observed Pax6 immu-noreactivity in some Olig2VP16-infected cells (9 � 3% at 7 dpi,n � 93; 20 � 2% at 14 dpi, n � 103; Fig. 3 H, H�, and I��),suggesting that Olig2 represses Pax6 also in this context, as in thedeveloping spinal cord (37) and the adult subependymal zone(39). Because the majority of Pax6-positive cells (57 � 14%)upon Olig2VP16 infection also expressed neuronal traits such asdcx immunoreactivity (Fig. 3 I–I���), the transcription factor Pax6may act as an important intermediate for the lineage changetoward a neuronal fate. In fact, transduction of cells with aPax6-containing retrovirus (22, 39) led to an earlier and furtherincrease in neurogenesis (19 � 2%, P � 0.007, n � 228, threemice, 7 dpi). However, only some of the dcx-positive cellsremained and differentiated into more mature neurons at laterstages after Olig2VP16 infection (Fig. 3K). As for glial cells, noCC1� oligodendrocytes differentiated amongst the Olig2VP16-infected cells compared to 10% of control virus injected cellsthat acquired this fate. Taken together, these data show that inan adult stab-lesioned cortex, endogenous neurogenesis can beevoked, and that a relief of the Olig2-mediated repression allowsthe induction of the neurogenic transcription factor Pax6,thereby instructing neurogenesis. Nevertheless, other strategiesare required to promote neuronal survival and furtherdifferentiation.

The Increase in Olig2 as a Pan-Lesion Phenomenon. To understand towhich extent the results described above have general implica-tions for adult brain injury, we examined other lesion models,comprising a further acute injury, the MCAO as a model forstroke (25), and a chronic injury paradigm with amyloid plaquedeposition in transgenic mice expressing the mutated forms ofAPP (27) and PS1 (28) under the Thy-1 promoter (Thy1-APPPS,see Materials and Methods). Despite the huge difference betweenthese injury models, the number of Olig2� cells increased in allof these injury paradigms (Fig. 4), 3-fold 7 days after MCAO(Fig. 4 A and C) and even 6-fold in the cortex of Thy1-APPPStransgenic mice compared to littermate controls (Fig. 4 B andD). Also, upon amyloid plaque deposition, the increase inOlig2� cells was observed at the mRNA level (Fig. 4 E and F).

However, a pronounced difference was detected in the celltype composition of Olig2� cells in the Thy1-APPPS cortexcompared to the stab-wound injury. The largest fraction ofOlig2� cells, the LM- cells in intact and stab-lesioned cortex, wasonly a minor component (10% of all of the Olig2� cells) in theThy1-APPPS cortex, mostly due to an increase in the proportionof Olig2� oligodendrocytes (40 � 8% versus 17 � 3% inwild-type cortices) and astrocytes (24 � 1% versus 11 � 1% ofwild types; 20.6 � 5.6% of Olig2� cells had GFAP). Thus, thehuge increase in Olig2� cells after amyloid deposition was

Fig. 3. Repression of Olig2 function after a stab wound. (A) The viral injectionexperimental design is illustrated: Animals were injected with either the GFP orthe Olig2VP16 virus 2 days after a stab wound and killed later. The micrograph (B)illustrates the distribution of GFP�-infected cells in the cortex 7 days after controlvirus injection (GM, gray matter; WM, white matter). (C–I) GFP� cells 7 days afterviral transduction (as specified in the images) stained for the antigens indicated.(F) Arrow points to a marker-negative infected cell. The micrographs (I–I���) showexamples of Pax6 and dcx coexpression in Olig2VP16-infected cells. The graphs inJ–L illustrate the phenotypes of virus-infected cells as indicated (three miceanalyzed for cell type identification at each time point; 100–200 analyzed cellsunless differently indicated in the text; dpi, days after injection). (Scale bars: B,200 �m; C–G, 20 �m; H, 40 �m; I–I���, 10 �m.)

18186 � www.pnas.org�cgi�doi�10.1073�pnas.0506535102 Buffo et al.

Dow

nloa

ded

by g

uest

on

Janu

ary

24, 2

021

caused by oligodendrocytes and other glial cells (90%), incontrast to the acute stab-wound containing mostly LM-�Olig2�cells (52%). Consistent with this difference, the proliferativereaction to injury was much less pronounced in the Thy1-APPPScortex with only a 1.7-fold increase in the number of BrdUrd-labeled cells (short pulse) as opposed to the 12-fold increase seenafter stab-wound injury (see above). Indeed, only a small numberof Olig2� cells in the Thy1-APPPS cortex were double-labeledwith BrdUrd after both 2 h (15 � 3% versus 45 � 2% in intactwild types) or 2 weeks of BrdUrd (7 � 4% versus 22 � 6% inintact wild types), indicating that proliferation of Olig2� cells iseven reduced upon amyloid deposition. Indeed, whereas thecortex of Thy1-APPPS mice CC1� oligodendrocytes comprisedthe majority of the increased Olig2� cells and virtually all ofthem displayed Olig2-positivity (92 � 8% in transgenic versus58 � 4% in controls), their total number was not significantlyincreased and they did not incorporate BrdUrd (Fig. 8, which ispublished as supporting information on the PNAS web site). Weconclude, therefore, that amyloid deposition induces transcrip-tional up-regulation of Olig2 most prominently in oligodendro-cytes and does not instruct Olig2� cell proliferation.

DiscussionThe present study shows that Olig2 induction in the adultneocortex is a common feature of both acute and chronic brain

lesions, whereas neurogenic fate determinants are not inducedupon injury. The increase in the number of Olig2� cells occurredupon various injury paradigms and in various regions of the CNS,such as the striatum, the spinal cord, and brain stem (40–42).Thus, the increase in the number of Olig2-positive cells is apan-lesion phenomenon that occurs throughout the CNS.

Diverse mechanisms seemingly lead to the increase in thenumber of Olig2� cells after lesion, such as transcriptional up-regulation and cell proliferation, although it cannot be excludedthat the recruitment of Olig2� progenitors�cells to the injury sitesalso contributes to this phenomenon. However, it is clear thatdistinct mechanisms are involved in the increase of the number ofOlig2� cells in distinct injury paradigms. Specific cell types (oli-godendrocytes) up-regulate Olig2 at the transcriptional level uponamyloid plaque deposition in the cortex of Thy1-APPPS mice,whereas Olig2� cells expand largely by proliferation upon acutestab-wound injury. These data reveal a striking difference in thereaction of distinct sets of glial cells to acute and chronic injury andprovide not only molecular insights into the brain reaction to injury,but also allowed the discovery of previously uncharacterized cellpopulations reacting to injury, such as the oligodendrocytes in theamyloid deposition model or the LM- cells reacting exclusively toacute injuries.

Strikingly, however, these diverse sets of cells activate thesame molecular pathway regulated by the transcription factorOlig2. Olig2 is expressed in diverse cell and precursor typesduring development (neuroepithelial cells, motoneuron precur-sors, and differentiating oligodendrocytes; refs. 33, 37, 38,43–45) and has recently been discovered to play an essential rolein transit-amplifying precursors in the adult subependymal zone(22, 39). Thus, our results could be interpreted such that Olig2may mediate some dedifferentiation aspects of glial cells uponinjury, because it is typically down-regulated in most mature celltypes (neurons, astrocytes, and even partially in oligodendro-cytes; refs. 33, 39, and 46). This interpretation is also consistentwith the decrease in LM- cells upon suppression of Olig2function, suggesting that these cells require Olig2 for the main-tenance of this un�dedifferentiated status. However, it is alsopossible that some precursors that either express or up-regulateOlig2 migrate to the injury site where they then multiply.Although we saw no evidence for such migration, only directviral-tracing experiments initiated before lesion will ultimatelyallow determining the origin of Olig2� cells.

Although high expression levels of Olig2 in precursor cells maywell be beneficial to expand their potential (see ref. 39), its laterdown-regulation is crucial to allow the progression along theneuronal lineage, as observed in the developing spinal cord and inthe adult olfactory bulb (39, 40, 46, 47). Indeed, here we could showthat antagonizing Olig2 function after stab lesion enabled a small,but significant, number of infected cells to generate neuroblasts.Because many antineurogenic factors are released upon acute braininjury (30, 48–50), the small number of neuroblasts is no surprise.However, these data show as a proof-of-principle that suppressionof antineurogenic determinants allows the progression of at leastsome precursors in the adult lesioned mammalian cortex toward aneurogenic fate. In fact, high levels of Olig2 may also be responsiblefor the exclusive glial fate of neurosphere cells transplanted into theinjured brain (22, 50–53).

Our data further unravel a key mechanism of Olig2 functionupon brain lesion, namely to repress neurogenic factors such asPax6, a transcription factor previously shown to be sufficient toconvert at least some astrocytes into neurons in vitro (17). Here,we could show that up-regulation of Pax6 by either Olig2VP16or Pax6 containing retroviral vectors is also sufficient in vivo toinstruct neurogenesis from cells that normally do not generateneurons. However, although in vitro the cells responding to Pax6were clearly astrocytes (17), the source of the newly generatedneurons is not clear in vivo. Whereas we could show that

Fig. 4. Increase in the number of Olig2-immunoreactive cells after focalischemia (MCAO) and in the Thy1-APPPS cortex. (A and B) Anti-Olig2 immu-nostaining of frontal sections of the cortical GM shows an increased numberof Olig2� cells after MCAO (A) and after amyloid plaque formation (B)compared to the control cortex (see Fig. 1A). Histograms in C and D display thecorresponding quantifications. hs, hours; dpl, days after lesion. In situ hybrid-ization reveals Olig2 mRNA up-regulation in the Thy1-APPPS (F) compared tothe wild-type cortex (E). (Scale bars: 100 �m.)

Buffo et al. PNAS � December 13, 2005 � vol. 102 � no. 50 � 18187

NEU

ROSC

IEN

CE

Dow

nloa

ded

by g

uest

on

Janu

ary

24, 2

021

neurogenesis can occur in the adult injured mammalian cortex,a major challenge is to improve survival and further differenti-ation of the newborn neurons because their numbers stronglydecreased until 30 days after viral injection. This decrease is mostlikely due to cell death, but viral construct silencing duringneuronal maturation cannot be excluded. Further steps, there-fore, are required to translate the initial neurogenesis towardlong-lasting neuronal repair. Taken together, this work identi-fied a central step toward endogenous repair by highlighting a

molecular mechanism interfering with endogenous neurogenesisfrom precursors reacting to brain injury.

We thank Ulrich Dirnagl for helpful suggestions on the manuscript,Charles Stiles (Harvard Medical School) for the anti-olig2 antibody, andBarbara Del Grande and Lana Polero for excellent secretarial help. Theanti-nestin antibody was obtained by the Developmental Studies Hy-bridoma Bank. This work was supported by the German ResearchFoundation (to M.G.) and by fellowships of The Von Humboldt Foun-dation and the Deutscher Akademischer Austauschdienst (to A.B.).

1. Doetsch, F., Caille, I., Lim, D. A., Garcia-Verdugo, J. M. & Alvarez-Buylla, A.(1999) Cell 97, 703–716.

2. Garcia, A. D., Doan, N. B., Imura, T., Bush, T. G. & Sofroniew, M. V. (2004)Nat. Neurosci. 7, 1233–1241.

3. Fawcett, J. W. & Asher, R. A. (1999) Brain Res. Bull. 49, 377–391.4. Ridet, J. L., Malhotra, S. K., Privat, A. & Gage, F. H. (1997) Trends Neurosci.

20, 570–577.5. Hartfuss, E., Galli, R., Heins, N. & Gotz, M. (2001) Dev. Biol. 229, 15–30.6. Malatesta, P., Hack, M. A., Hartfuss, E., Kettenmann, H., Klinkert, W.,

Kirchhoff, F. & Gotz, M. (2003) Neuron 37, 751–764.7. Mori, T., Buffo, A. & Gotz, M. (2005) in Current Topics in Developmental

Biology, ed. Schatted, G. (Elsevier Life Sciences, San Diego) 69, 67–99.8. Doetsch, F., Garcia-Verdugo, J. M. & Alvarez-Buylla, A. (1999) Proc. Natl.

Acad. Sci. USA 96, 11619–11624.9. Gates, M. A., Thomas, L. B., Howard, E. M., Laywell, E. D., Sajin, B., Faissner,

A., Gotz, B., Silver, J. & Steindler, D. A. (1995) J. Comp. Neurol. 361, 249–266.10. Temple, S. & Alvarez-Buylla, A. (1999) Curr. Opin. Neurobiol. 9, 135–141.11. Seri, B., Garcia-Verdugo, J. M., McEwen, B. S. & Alvarez-Buylla, A. (2001)

J. Neurosci. 21, 7153–7160.12. Seri, B., Garcia-Verdugo, J. M., Collado-Morente, L., McEwen, B. S. &

Alvarez-Buylla, A. (2004) J. Comp. Neurol. 478, 359–378.13. Doetsch, F., Petreanu, L., Caille, I., Garcia-Verdugo, J. M. & Alvarez-Buylla,

A. (2002) Neuron 36, 1021–1034.14. Malatesta, P., Hartfuss, E. & Gotz, M. (2000) Development (Cambridge, U.K.)

127, 5253–5263.15. Noctor, S. C., Flint, A. C., Weissman, T. A., Dammerman, R. S. & Kriegstein,

A. R. (2001) Nature 409, 714–720.16. Miyata, T., Kawaguchi, A., Okano, H. & Ogawa, M. (2001) Neuron 31, 727–741.17. Heins, N., Malatesta, P., Cecconi, F., Nakafuku, M., Tucker, K. L., Hack, M. A.,

Chapouton, P., Barde, Y. A. & Gotz, M. (2002) Nat. Neurosci. 5, 308–315.18. Laywell, E. D., Rakic, P., Kukekov, V. G., Holland, E. C. & Steindler, D. A.

(2000) Proc. Natl. Acad. Sci. USA 97, 13883–13888.19. Belachew, S., Chittajallu, R., Aguirre, A. A., Yuan, X., Kirby, M., Anderson,

S. & Gallo, V. (2003) J. Cell Biol. 161, 169–186.20. Aguirre, A. A., Chittajallu, R., Belachew, S. & Gallo, V. (2004) J. Cell Biol. 165,

575–589.21. Alonso, G. (2005) Glia 49, 318–338.22. Hack, M. A., Sugimori, M., Lundberg, C., Nakafuku, M. & Gotz, M. (2004)

Mol. Cell Neurosci. 25, 664–678.23. Parras, C. M., Galli, R., Britz, O., Soares, S., Galichet, C., Battiste, J., Johnson,

J. E., Nakafuku, M., Vescovi, A. & Guillemot, F. (2004) EMBO J. 23,4495–4505.

24. Toresson, H., Potter, S. S. & Campbell, K. (2000) Development (Cambridge,U.K.) 127, 4361–4371.

25. Vosko, M. R., Burggraff, D., Liebetrau, M., Wunderlich, N., Jager, G., Groger,M., Plesnila, N. & Hamann, G. F. Neurol. Res. 27, in press.

26. Hata, R., Mies, G., Wiessner, C., Fritze, K., Hesselbarth, D., Brinker, G. &Hossmann, K. A. (1998) J. Cereb. Blood Flow Metab. 18, 367–375.

27. Bondolfi, L., Calhoun, M., Ermini, F., Kuhn, H. G., Wiederhold, K. H., Walker,L., Staufenbiel, M. & Jucker, M. (2002) J. Neurosci. 22, 515–522.

28. Moehlmann, T., Winkler, E., Xia, X., Edbauer, D., Murrell, J., Capell, A.,Kaether, C., Zheng, H., Ghetti, B., Haass, C. & Steiner, H. (2002) Proc. Natl.Acad. Sci. USA 99, 8025–8030.

29. Nimmerjahn, A., Kirchhoff, F. & Helmchen, F. (2005) Science 308, 1314–1318.30. Yamamoto, S., Nagao, M., Sugimori, M., Kosako, H., Nakatomi, H.,

Yamamoto, N., Takebayashi, H., Nabeshima, Y., Kitamura, T., Weinmaster,G., et al. (2001) J. Neurosci. 21, 9814–9823.

31. Lu, Q. R., Sun, T., Zhu, Z., Ma, N., Garcia, M., Stiles, C. D. & Rowitch, D. H.(2002) Cell 109, 75–86.

32. Arnett, H. A., Fancy, S. P., Alberta, J. A., Zhao, C., Plant, S. R., Kaing, S.,Raine, C. S., Rowitch, D. H., Franklin, R. J. & Stiles, C. D. (2004) Science 306,2111–2115.

33. Liu, Y. & Rao, M. S. (2004) Glia 45, 67–74.34. Stolt, C. C., Lommes, P., Friedrich, R. P. & Wegner, M. (2004) Development

(Cambridge, U.K.) 131, 2349–2358.35. Price, J. (1987) Development (Cambridge, U.K.) 101, 409–419.36. Hajihosseini, M., Iavachev, L. & Price, J. (1993) EMBO J. 12, 4969–4974.37. Mizuguchi, R., Sugimori, M., Takebayashi, H., Kosako, H., Nagao, M.,

Yoshida, S., Nabeshima, Y., Shimamura, K. & Nakafuku, M. (2001) Neuron 31,757–771.

38. Novitch, B. G., Chen, A. I. & Jessell, T. M. (2001) Neuron 31, 773–789.39. Hack, M. A., Saghatelyan, A., de Chevigny, A., Pfeifer, A., Ashery-Padan, R.,

Lledo, P. M. & Gotz, M. (2005) Nat. Neurosci. 8, 865–872.40. Fancy, S. P., Zhao, C. & Franklin, R. J. (2004) Mol. Cell Neurosci. 27, 247–254.41. Han, S. S., Liu, Y., Tyler-Polsz, C., Rao, M. S. & Fischer, I. (2004) Glia 45, 1–16.42. Talbott, J. F., Loy, D. N., Liu, Y., Qiu, M. S., Bunge, M. B., Rao, M. S. &

Whittemore, S. R. (2005) Exp. Neurol. 192, 11–24.43. Takebayashi, H., Yoshida, S., Sugimori, M., Kosako, H., Kominami, R.,

Nakafuku, M. & Nabeshima, Y. (2000) Mech. Dev. 99, 143–148.44. Takebayashi, H., Nabeshima, Y., Yoshida, S., Chisaka, O., Ikenaka, K. &

Nabeshima, Y. (2002) Curr. Biol. 12, 1157–1163.45. Zhou, Q. & Anderson, D. J. (2002) Cell 109, 61–73.46. Lee, S. K., Lee, B., Ruiz, E. C. & Pfaff, S. L. (2005) Genes Dev. 19, 282–294.47. Lu, Q. R., Cai, L., Rowitch, D., Cepko, C. L. & Stiles, C. D. (2001) Nat.

Neurosci. 4, 973–974.48. Monje, M. L., Toda, H. & Palmer, T. D. (2003) Science 302, 1760–1765.49. Ekdahl, C. T., Claasen, J.-H., Bonde, S., Kokaia, Z. & Lindvall, O. (2003) Proc.

Natl. Acad. Sci. USA 100, 13632–13637.50. Lim, D. A., Tramontin, A. D., Trevejo, J. M., Herrera, D. G., Garcia-Verdugo,

J. M. & Alvarez-Buylla, A. (2000) Neuron 28, 713–726.51. Cao, Q., Benton, R. L. & Whittemore, S. R. (2002) J. Neurosci. Res. 68, 501–510.52. Eriksson, C., Bjorklund, A. & Wictorin, K. (2003) Exp. Neurol. 184, 615–635.53. Hofstetter, C. P., Holmstrom, N. A., Lilja, J. A., Schweinhardt, P., Hao, J.,

Spenger, C., Wiesenfeld-Hallin, Z., Kurpad, S. N., Frisen, J. & Olson, L. (2005)Nat. Neurosci. 8, 346–353.

18188 � www.pnas.org�cgi�doi�10.1073�pnas.0506535102 Buffo et al.

Dow

nloa

ded

by g

uest

on

Janu

ary

24, 2

021